Mismatched base pairing in DNA duplexes may arise due to errors introduced during DNA replication (Echols and Goodman (1991) Annu. Rev. Biochem. 60:477-511; Kornberg and Baker (1991) DNA replication, W. H. Freeman & Co., New York), heteroduplex formation during homologous recombination (Holliday (1964) Genet Res. 5:282-304; Petes and Hill (1988) Annu. Rev. Genet. 22:147-168), as a consequence of mutation and by enzymatic modification of DNA such as deamination of 5-methylcytosine. Such mismatches can lead to genome instability. Therefore, all living systems have evolved specialized pathways to repair specific mismatches which are somewhat different than other DNA repair mechanisms such as base excision repair and nucleotide excision repair (Wildenberg and Messelson (1975) Proc. Natl. Acad. Sci. USA 72:2202-2206; Wagner and Messelson (1996) Proc. Natl. Acad. Sci. USA 73:4136-4139; Radman and Wagner (1986) Annu. Rev. Genet. 20:523-538; Freidberg (1985) DNA Repair, W. H. Freeman & Co., New York). Early studies in prokaryotic systems, especially Escherichia coli, led to the identification of one of these pathways, called the long-patch repair system or the methyl-directed mismatch-repair system (Radman and Wagner (1986) Annu. Rev. Genet. 20:523-538). This pathway exhibits rather broad specificities for repairing mismatches generated during DNA biosynthesis as well as recombination. Several genes essential for the methyl-directed mismatch repair have been identified in E. Coli. Primary among these are mutS, mutL, mutH, UvrD, and the Dam methyltransferase and exonuclease genes (Freidberg (1985) DNA Repair, W. H. Freeman & Co., New York).
Genetic evidence for the existence of the mismatch-repair pathways in eukaryotes has been around since the late 1960s (Emerson (1969) Genetic Organization Caspari & Ravin, eds., Academic Press, New York, pp. 267-360). However, it was not until the early 1990s, following the first biochemical evidence for the repair activity in a eukaryote (Muster-Nassal and Kolodner (1986) Proc. Natl Acad. Sci. USA 83:7618-7622) and the isolation and characterization of yeast mutS homologues, Mshl (Reenan and Kolodner (1992) Genetics 132:963-973), MSH2 (Reenan and Kolodner (1992) Genetics 132:975-985) and Msh3 (New et al. (1993) Mol. Gen. Genet. 239:97-108), that the existence of a mismatch-repair pathways in eukaryotes was clearly established. Subsequently, several eukaryotic Msh genes have been cloned and characterized (Nickoloff and Hoeskstra (1998) DNA Damage and Repair, vols. I-II, Humana Press, New York). Extensive and careful biochemical studies over the past decade have revealed that the gene products (denoted by MSH1, MSH2 etc.) of individual Msh gene family members exhibit remarkable specificity in their ability to participate in different biological processes. Thus, in yeast, MSH1 is primarily responsible for mitochondrial DNA repair, MSH2, MSH3, and MSH6 are involved in base mismatch repair and in modulating recombination, whereas MSH4 and MSH5 and are involved in modulating recombination. Precisely how MSH2, MSH3, and MSH6 participate in recombination has not yet been determined. It has been proposed that in addition to their mismatch-repair activity, these gene products interact with other cellular components involved in resolution of the Holliday junction (Nickoloff and Hoeskstra (1998) DNA Damage and Repair, vols. I-II, Humana Press, New York).
Interestingly, in a recent study with mammalian cells, mismatch repair has been shown to have an anti-recombinational effect. Thus, in a mouse msh2 cell line, target integration of a plasmid DNA at the Rb locus was increased 50-fold. Furthermore, MSH2 and Msh3 homologues are known to be involved in gene targeting and gene modification processes (deWind et al. (1995) Cell 82:321-330). The deWind reference, as well as the Abuin et al. ((2000) Cellular Biol 20:149-157), disclose that MSH2 deficiency increases the recombination frequency between non-identical DNA substrates. The anti-recombination effect is only observed with non-identical DNAs; recombination between identical DNA substrates is unaffected in msh2 lines. This is likely because pairing of homologous DNA sequences does not lead to DNA mismatches.
A combination of factors appears to render plant genomes highly susceptible to mutation. Complex genomes of higher plants contain large numbers of putative mutational hotspots, such as microsatellites, repeated elements and 5-methylcytosine. In addition, unlike many other multicellular organisms, plant germ cells are derived from somatic progenitors that have undergone many cell divisions. High DNA replication fidelity is crucial to the faithful transmission of genetic information to subsequent plant generations. The DNA mismatch-repair system plays a crucial role in maintaining the integrity of the genome. Mismatch-repair activities identify and catalyze the repair of DNA polymerase errors and base-pair mismatches and act to restrict recombination between non-homologous DNA sequences. The proofreading and anti-recombination functions of mismatch-repair activities likely play a key role in the fitness of subsequent plant generations.
The methyl-directed mismatch-repair system of E. coli is well characterized. For review, see Modrich and Lahue ((1996) Annu. Rev. Biochem. 65:101-133). In brief, the key components in E. coli mismatch repair are: MutS, which interacts directly with mismatched DNA; and MutL, which, through its interaction with MutS, activates the MutH endonuclease. Upon activation, MutH endonuclease introduces a nick in the unmethylated DNA flanking the site of the base-pair mismatch at a hemi-methylated GATC site. The nicked strand is then degraded through the site of the mismatch and the degraded sequences are resynthesized and ligated.
Eukaryotes encode a family of MutS orthologs or homologs, known as Msh. Mismatch recognition in eukaryotes is accomplished by a heterodimer of MSH proteins, depending upon the type of mismatch. Heterodimers of MSH2 and MSH3 recognize insertion mismtaches and DNA loops, while heterodimers of MSH2 and MSH6 interact preferentially with base-pair mismatches and single base insertions. (Marsischky et al. (1996) Genes Devel. 10:407-420) MSH2 is the key component in mismatch recognition, because it is required to initiate correction of any sort of mismatch. Biochemical and genetic studies in E. coli have demonstrated an antirecombination activity associated with homologs of MutS (Rayssiguier et al. (1989) Nature 342:396-401). The role of MSH2 in preventing recombination between partially homologous (homeologous) sequences has also been established in S. cerevisiae (Alani et al. (1994) Genetics. 137:19-39).
While much is known about the biochemical nature of DNA mismatch repair in bacterial, yeast, and mammalian systems, very little is known about the corresponding repair pathways in plants. MutS homolog genes have been identified in a number of plant species, including Arabidopsis, maize and wheat, but the contributions of these proteins to genome stability and DNA proofreading has not been established.